The goal of this proposal is to elucidate the folding mechanism of two o_-(3proteins, the N- terminal domain of the ribosomal protein L9 (NTL9) and the C-terminal domain of L9 (CTL9). NTL9 is one of the simpler examples of an important common class of layered sheet-helix structures. CTL9 contains an interesting mixed parallel anti-parallel I_-sheet. Key questions that will be addressed include the nature of the denatured state and the effects of denatured state structure on folding and stability. How broad or narrow is the transition state? What is the detailed nature of the transition state for folding? Does mutational analysis provide an accurate picture of the transition state? The insights provided by this work are expected to have significant impact on biotechnology, biophysics and basic biomedicine. Electrostatic interactions play a key role in the denatured state of NTL9. Analysis of a set of mutants will determine their origin. The effect of modulating these interactions on folding will be determined by combining kinetic studies with mutational analysis. A set of kinetic folding experiments with variants containing natural and unnatural aminoacids will be carried out to provide information on the details of sidechain interactions in the transition state. Isotope effect experiments will provide key information about backbone structure in the transition state. Collaborative work will allow us to compare our results with the predictions of computational studies. These experiments will provide a unique high-resolution view of the transition state for folding. The folding of CTL9 will be investigated. No folding studies of this type of motif have been carried out. The role of buried polar interactions in the folding of CTL9 will be elucidated and the properties of the denatured state examined by mutagenesis and pH dependent studies. Mutational analysis will be used to map out the transition state for folding. The specific aims are 1) To elucidate electrostatic interactions in the denatured state of NTL9 2) To determine their role in folding 3) To develop a high resolution view of the transition state for the folding of NTL9 4) To develop a detailed picture of the folding of CTL9.